Lipid A is the active moiety of lipopolysaccharide (LPS, endotoxin), a bacterial product important in the pathogenesis of Gram-negative sepsis. Lipid A activates phagocytes through a defined receptor system. While LBP and CD14 enhance LPS responses, two proteins are obligatory for signal transduction: Toll-like receptor (TLR) 4 and MD-2. This multimeric receptor utilizes all four TLR-adapter molecules: MyD88, Mal, TRAM and TRIF, giving TLR4/MD-2 a complexity that is unrivaled by other TLRs. In the past, we focused on three aspects of TLR4 activation: first, what we could learn about TLR4 by comparing it to other TLRs. Second, how MD-2 binds LPS. Third, how Mal and TRAM interact with TLR4. In the next funding period, our goal is to determine how TLR4 achieves an active state (i.e., one that transduces signals) and to better delineate how TIR domain containing adapters modulate gene expression. We will focus on techniques with which we have become expert: the creation of novel cell lines, the production of recombinant TLRs, confocal and electron microscopy, microarray analysis and the exploitation of transgenic mice. In the Aim 1, we propose to identify what constitutes an active TLR4/MD-2 receptor complex. We will focus on conformational changes induced by ligands, as well as the dimerization status of TLR4/MD-2. In Aim 2, we will establish the rules for adapter molecule engagement. We will perform a much-overdue global analysis of downstream signaling events specifically mediated by adapter molecules by RNA profiling LPS-stimulated macrophages from TLR adapter knockout mice. These studies will be validated by real time PCR, and test the canonical model (which we hypothesize to be flawed) that MyD88/Mal activates proinflammatory genes while the remained of gene expression is subserved via TRAM/TRIF. We will analyze the functions of known polymorphisms associated with Mal and MyD88 in macrophage cell lines derived from KO mice, and determine how these polymorphisms influence adapter molecule recruitment. In Aim 3, we will focus on MD-2, the binding portion of TLR4/MD-2, which we have purified to homogeneity in its monomeric (active) state. We will use biophysical approaches (e.g., circular dichroism, FLIM) to assess ligand-induced conformational changes in MD-2 and TLR4. We will combine mutagenesis and forward genetic screening to identify mutants of MD-2 that are constitutively active, and determine their structure/function both empirically and in silico by building on the recently resolved crystal for MD-2. Finally, virtually nothing is known about the regulation and role of MD-2 during inflammatory states in vivo. We will expand upon the limited number of reagents available for MD-2 by generating anti-mouse MD-2 mAbs. We also propose to engineer a transgenic mouse in which GFP is under the control of the MD-2 promoter and natural MD-2 will be epitope tagged with FLAG. We will determine which cells produce MD-2 and how much is produced during inflammation. Ultimately, we believe that an improved understanding of TLR4/MD-2 biology will lead to an amelioration of the morbidity and mortality of sepsis. Toll-like receptors (TLRs) are molecules on white blood cells that recognize microbes and lead to immune defense and inflammation. There are many diseases caused by TLR activation, but none is more deadly than LPS (lipid A) induced sepsis, which is caused by activation of the TLR4/MD-2 receptor complex. We propose to learn how the TLR4/MD-2 receptor is activated by LPS (lipid A), in order that the high mortality and morbidity of sepsis can be ameliorated.